A novel sulfatase, specific for the 3-0 sulfate ester of methyl-2-deoxy-2-sulfamino-alpha-D-glucopyranoside 3-sulfate, has been purified approximately 70-fold from human urine. By means of definitively synthesized 35S-labeled glucosamine derivatives, it has been established that the enzyme a) is specific for the 3-0 sulfate ester and b) requires that the adjacent amino group be sulfated rather than free or acetylated. A disulfated glucosamine residue corresponding to the synthetic enzyme substrate is not known to exist in any natural substance. Among the glycosaminoglycans, only heparin and heparan sulfate can readily accomodate a 3-0 sulfated sulfaminoglucose moiety. Using the purified 3-0 sulfatase as a structural probe, evidence has been obtained indicating that both 3-0 sulfated and 3,6 disulfated residues are uniquely present in heparin fragments having a high affinity for antithrombin. The presence of 3-0 sulfated glucosamine residues is an important new modification in the accepted structure of heparin; such residues may play a key role in the mechanism of inhibition of blood coagulation by the heparin-antithrombin complex.